Methods for magnetic immobilization and manipulation of cells

ABSTRACT

Microbiological entities, such as cells or microbes, are immobilized in a single-file linear array for optical analysis. Colloidal magnetic particles having a binding agent, such as ligand for attachment with a corresponding receptor, are bound to the entities suspended in a fluid medium. The fluid medium is placed into a vessel having a ferromagnetic capture structure including an elongated linear collection surface with a diameter less than that of the microbiological species of interest. The vessel is placed into a magnetic field for inducing a magnetic gradient in a region along the collection surface of the ferromagnetic capture structure. The magnetically-labeled entities are attracted toward the collection surface and immobilized thereon in a linear array. Microscopic optical and fluorescence observations, and sequential chemical reactions and mechanical manipulations may be performed on the line of immobilized entities. Methods of analysis for blood and immune cells, such as leukocytes and lymphocytes, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 08/424,271, filedunder the Patent Cooperation Treaty on Apr. 24, 1995, now abandonedwhich is a continuation-in-part of U.S. application Ser. No. 07/976,476,filed Nov. 16, 1992, now abandoned, which was a continuation-in-part ofU.S. application Ser. No. 07/588,662, filed Sep. 26, 1990, now U.S. Pat.No. 5,200,084.

FIELD OF THE INVENTION

This invention relates to the immobilization and manipulation ofmicroscopic biological entities. More particularly, the presentinvention relates to magnetic labelling and immobilization ofmicroscopic biological entities within an apparatus having anobservation path for allowing observation and manipulation of suchentities suspended within a fluid medium.

BACKGROUND

Many biological techniques such as are employed in biotechnology,microbiology, clinical diagnostics and treatment, in vitrofertilization, hematology and pathology, require such processes asidentification, separation, culturing, or manipulation of a targetentity such as a type of cell or microbe within a fluid medium such asblood, other bodily fluids, culture fluids or samples from theenvironment. It is often desirable to retain viability of the targetentity or to culture the target entity.

Identification techniques typically involve labelling the target entitywith a reagent which can be detected according to a characteristicproperty. Entities which can be viewed optically such as cells orcertain microbes, may be identified using fluorescent MAb's or stainingreagents specific to certain classes of cells or microbes. When suchidentification is done manually or mechanically, as by microscopy,multiple operations involving incubations and washing steps to removeexcess labelling reagent are often performed. For example, in the usualmethod used to identify a subset of T-lymphocytes, such as T-HelperCells or CD4-positive cells, a mixture of peripheral blood lymphocytesis incubated with a fluorescent M0Ab directed to CD4-positive cells. TheMAb is then given sufficient incubation time to react with theCD4-positive cells. The CD4-positive cells are then washed usingmultiple centrifugations and can then readily be identified byfluorescent microscopy.

In the practice of manual fluorescent labelling methods employing afluorescent microscope, direct labeling with MAb's is often impracticaldue to the expense of obtaining a cell-specific fluorescent Mab andbecause of reduced signal availability. Thus the technique of indirectanalysis is common. During indirect analysis, the target entities arefirst labeled with a specific non-fluorescent MAb. Excess Mab is washedaway. Then, a fluorescent-labeled second reagent such asfluorescent-labeled goat anti-mouse antibody is added to the medium. Themedium is allowed to incubate to allow the labelled second reagent tobind with the non-fluorescent MAb and then excess reagent is removed.The target entities may then be identified due to the attachment of thefluorescent secondary reagent to the non-fluorescent biospecific MAb.Such methods are time-consuming, costly, and require considerablequantities of reagents. Moreover, as the number of operations employedin such identification processes increases, a greater number of targetentities are lost or killed. Accurate microbial analyses employing suchmethodologies are difficult to achieve because of the small numbers oftarget entities involved as well as the difficulty of washing awayunbound labeling agents. Other methodologies such as flow cytometry(fluorescent activated cell sorting) or field flow fractionation can beused for such analysis and in some instances require fewermanipulations. These other methods, however, require expensiveequipment, highly trained personnel and typically can only analyze orseparate one sample at a time.

Manipulation of target entities required by other biological techniquesmay also involve such processes as insertion of genetic material,organelles, subcellular components, viruses, or other foreign materialsor bodies into the target entities. Inserted materials can be labeledprior to insertion so that effects and movements of these materials canbe studied during incubation of the medium. In techniques such astransfection, or in vitro fertilization mechanical probes or arms areoften used to hold the target entities. Such mechanical holding methodstend to obscure or damage the target entities.

It would be desirable in such biotechnical procedures as have beendiscussed to provide devices and methods for precise non-destructiveimmobilization and manipulation of specific target entities in aninexpensive and rapid manner.

Magnetic colloids having particles coated with biospecific compoundswhich attach to target entities are known to be useful in certainbiospecific separation techniques. Reaction rates between such colloidalparticles and target entities can be relatively rapid due to fastkinetic activity of the particles and sufficiently large areas ofexposed reactant coatings. Magnetic particles in the range of 0.7 to 1.5microns have been described in U.S. Pat. Nos. 3,790,518; 4,018,886;4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain ofthese particles are disclosed to be useful solid supports forimmunologic reagents, having reasonably good suspension characteristicswhen mildly agitated. Magnetic particle suspensions presently incommercial use tend to flocculate in time and must be resuspended byagitation prior to use. Such agitation adds another step to any processemploying such reagents.

Small magnetic particles, such as those mentioned above, generally fallinto two broad categories: particles that are permanently magnetized;and particles that become magnetic when subjected to a magnetic field.The latter particles are referred to herein as magnetically-responsiveparticles. Materials displaying magnetically-responsive behavior aresometimes described as superparamagnetic. However, certain ferromagneticmaterials such as magnetic iron oxide crystals, behave in amagnetically-responsive manner when the crystals are less than about 30nm in diameter. Larger crystals of ferromagnetic materials, by contrast,retain permanent magnet characteristics after exposure to a magneticfield and tend to aggregate thereafter. The properties of such particlesare described in P. Robinson et al., Biotech Bioeng. XV:603-06 (1973).

Magnetically-responsive colloidal magnetite is disclosed in U.S. Pat.No. 4,795,698 to Owen et al., which relates to polymer-coated,sub-micron size magnetite particles that behave as true colloids.Several devices are known which are used to separate magnetic particlesfrom colloidal suspensions. Examples of such devices are magneticseparators such as the MAIA Magnetic Separator manufactured by SeronoDiagnostics, Norwell, Mass.; the DYNAL MPC-1 manufactured by DYNAL,Inc., Great Neck, N.Y.; and the BioMag Separator, manufactured byAdvanced Magnetics, Inc., Cambridge, Massachusetts. A similar magneticseparator, manufactured by ciba-corning Medical Diagnostics, Wampole,Mass. is provided with rows of bar magnets arranged in parallel andlocated at the base of the separator. This device accommodates 60 testtubes, with the closed end of each test tube fitting into a recessbetween two of the bar magnets.

The above-described magnetic separators have the disadvantage that themagnetic particles and other impurities tend to form several layers onthe inner surface of the sample container where they become entrappedand are difficult to remove even with vigorous washing. These separatorsare also not capable of establishing monolayers of target entities formicroscopic analysis or manipulation.

Separation of magnetically-responsive particles within colloidalsuspensions requires high gradient field separation techniques such asare described in R.R. Oder, IEEE Trans. Magnetics, 12:428-35 (1976); C.Owen and P. Liberti, Cell Separation: Methods and Selected Applications,Vol. 5, Pretlow and Pretlow eds., Academic Press, N.Y., (1986). Gradientfields normally used to filter such materials generate relatively largemagnetic forces. Another useful technique for performing magneticseparation of colloidal magnetic particles from a test medium by theaddition of agglomerating agents is disclosed in and commonly-owned U.S.Pat. No. 5,108,933 issued Apr. 28, 1992.

A commercially available high gradient magnetic separator, the MACSdevice made by Miltenyi Biotec GmbH, Gladback, West Germany, employs acolumn filled with a non-rigid steel wool matrix in cooperation with apermanent magnet. In operation, the enhanced magnetic field gradientproduced in the vicinity of the steel wool matrix attracts and retainsthe magnetic particles while the non-magnetic components of the testmedium pass through the column. It has been found that the steel woolmatrix of such prior art high-gradient magnetic separation (HGMS)devices often causes non-specific entrapment of biological entitiesother than the target entities. The entrapped non-magnetic componentscannot be removed completely without extensive washing and resuspensionof the particles bearing the target substance. Moreover, the sizes ofthe columns in many of the prior art HGMS devices require substantialvolumes of test media, which poses an impediment to their use inperforming various useful laboratory-scale separations. In addition, thesteel wool matrix may damage sensitive cell types.

Although HGMS affords certain advantages in performing medical orbiological analyses based on biospecific affinity reactions involvingcolloidal magnetic particles, the systems developed to date are notparticularly suited for immobilization and micromanipulation. Forexample, collection of microscopic entities upon an irregular structuresuch as steel wool is not conducive to microscopic observation whereinit is desirable to maintain the subject of interest in the focal planeof a microscope. Furthermore, the convoluted surface of the steel woolwould obscure observation of the collected entities. Accordingly, itwould be desirable to provide an HGMS apparatus for immobilization andmicromanipulation of target entities which is of relatively simpleconstruction and operation and yet maximizes magnetic field gradients,so as to be of practical utility in conducting various laboratory-scaleseparations and micromanipulations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention an apparatus isprovided for immobilizing selected microscopic biological entitieswithin a non-magnetic vessel containing a fluid medium. A colloidalsuspension of submicroscopic magnetically-responsive particles having abiospecific coating for binding with the selected entities is introducedinto the medium, the particles become attached to the selected entities,and are subsequently attracted to a collection structure within oradjacent to the interior of the vessel that produces an intense magneticfield gradient upon application of an external magnetic field by amagnet structure having poles in opposition on either side of thevessel. Alternatively, non-specific binding to some or all of theentities can be employed.

The spatial distribution in which entities are collected is related tothe shape of the ferromagnetic collection structure and theconcentration of magnetically-responsive particles in the fluid medium.The ferromagnetic collection structure provides a collection surfaceupon which the target entities are immobilized in an orderly manner sothat observation and/or manipulation of the immobilized entities iseasily accomplished relative to known techniques.

Ferromagnetic collection structures having sufficiently high curvatures,a multi-stranded construction, or sharp edges are capable of producingsubstantially monolayered one-dimensional spatial distributions ofimmobilized entities. Additionally, the concentration ofmagnetically-responsive particles may be selected to facilitate theformation of such a monolayer, for example by controlling theconcentration of magnetically-responsive particles suitable to bind withtarget entities such that the surface area of the bound target entitiesis commensurate with the surface area of the ferromagnetic collectionstructure or other collection surface within the magnet separator.

A non-magnetic vessel for use in a magnetic immobilization apparatus ofthe invention preferably has an observation path which allowsmicroscopic observation of the immobilized entities. According to oneaspect of the invention, the observation path includes at least one openor transparent aperture or surface in the non-magnetic vessel forallowing substantially unobscured microscopic observation and physicalmanipulation, such as microsurgery, of the immobilized entities.According to another aspect of the invention, the vessel may include atleast one surface or aperture that is transparent to at least a portionof the electromagnetic spectrum required for a particular observationaltechnique. The observation path is oriented relative to the collectionstructure to allow substantially unobscured observation of theimmobilize; entities. The collection structure is preferably arranged tomaintain a plurality of the immobilized entities within an orderly arrayintersecting the observation path, such as within a focal plane of amicroscope.

A vessel may further have ports which allow a flow of liquid reagentsthrough the vessel as may be desired in, for example, sequentialreaction techniques. The flowthrough vessel also facilitates observationof the immobilized entities by allowing the test medium to be flushed orrinsed. Such flushing or rinsing may be employed to remove an opaquetest medium from the vessel while leaving behind the immobilizedentities or substances. Furthermore, flushing or rinsing of the testmedium allows control of the duration of the separation process so thatthe test medium may be removed from the vessel after having been placedin the magnetic field for a period of time sufficient for formation of amonolayered array of immobilized entities upon the collection structureand within the observation path.

In accordance with another aspect of the invention, an apparatus mayprovide for lateral translation or concentration of immobilized entitiesby the use of a transverse obstruction, or shoulder, which concentratesimmobilized entities in the vicinity of the obstruction, or shoulder, asthe shoulder is moved laterally along the axis of the collectionstructure within or out of the field confined to the region between theopposing poles.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofa preferred embodiment of the present invention, will be betterunderstood when read in conjunction with the appended drawings, inwhich:

FIGS. 1A-1C are exploded views of embodiments of a magnetic mobilizationapparatus;

FIGS. 2A and 2B are fragmentary perspective views of ferromagneticcollecting elements suitable for use within the embodiments of FIGS.1A-1C;

FIGS. 3A-3D are perspective views of central troughs suitable forvessels within the embodiments of FIGS. 1A-1C;

FIGS. 4A and 4B are overhead perspective views of devices for collectingand concentrating target entities; and

FIG. 5 is a schematic diagram of an arrangement for observing andmanipulating entities collected within a magnetic immobilizationapparatus.

DETAILED DESCRIPTION

In accordance with the present invention, target entities within a fluidmedium are immobilized for observation, analysis, or manipulation. Suchtarget entities as may be immobilized hereby include cellular entitiesand subcellular entities.

Immobilization of a target entity is accomplished by magnetic labellingand subsequent collection of the target entity. Magnetic labelling of atarget entity is achieved by the use of a colloidal suspension ofmagnetically-responsive particles having a coating which includes anattachment agent such as a ligand selected to bind to a correspondingreceptor of the target entity. The colloidal suspension of magneticallyresponsive particles is mixed with the fluid medium containing thetarget entities to form a test medium. Such mixing can be carried outeither prior to or after the fluid medium is introduced into anon-magnetic vessel. A magnetic field is then applied to the vessel.

Upon application of a magnetic field to the non-magnetic vessel, themagnetically-labelled target entities tend to migrate toward and adhereto a collection structure or magnetic flux intensifier such as aferromagnetic wire supported within or adjacent to a vessel containingthe test medium. The strength of the magnetic field, preferably betweenabout 4, kGauss and 15 kGauss, influences the time required to effectcollection. Additionally, the impact and binding forces experienced bythe target entities upon the collection structure are determined in partby the strength of the applied magnetic field. The long-range attractivemagnetic force acting upon the target entities within the test medium ismainly due to the size of the collection structure and the bulkpermeability of the material from which the collection structure ismade. The short-range binding forces between the collection structureand the collected target entities is mainly due to the surface geometryof the collection structure.

The preferred magnetic particles for use in carrying out this inventionare particles that behave as true colloids. Such particles arecharacterized by their sub-micron particle size, which is generally lessthan about 200 nanometers (nm) (0.20 microns), and their stability togravitational separation from solution for extended periods of time.Such small particles facilitate observation of the target entities viaoptical microscopy since the particles are significantly smaller thanboth the target entities and the wavelength range of visible light.Suitable magnetically-responsive particulate materials are composed of acrystalline core of superparamagnetic material surrounded by moleculeswhich may be physically absorbed or covalently attached to the magneticcore and which confer stabilizing colloidal properties. The size of thecolloidal particles is sufficiently small that they do not contain acomplete magnetic domain, and their Brownian energy exceeds theirmagnetic moment. As a consequenced, North Pole, South Pole alignment ofthe particles and subsequent mutual attraction/repulsion of thesecolloidal magnetic particles does not appear to occur even in moderatelystrong magnetic fields. The lack of such particle-particle interactionscontributes to the stability of the colloidal suspension. Accordingly,colloidal magnetic particles are not readily separable from solution assuch even with powerful electromagnets. Rather, a magnetic gradient isrequired within the test medium in which the particles are suspended inorder to achieve separation of the discrete particles.

Magnetic particles having the above-described properties can be preparedas described in U.S. Pat. No. 4,795,698, the entire disclosure of whichis incorporated by reference in the present specification, as if setforth herein in full.

For immobilization of cellular target entities, the test medium istypically prepared from appropriately prepared fluids, for example, bodyfluids such as blood, urine, sputum or secretions. It is preferable toadd the colloidal magnetic particles to the test medium in a buffersolution. A suitable buffer solution for this purpose comprises amixture of 5% bovine serum albumin ("BSA") and 95% of a biocompatiblephosphate salt solution, optionally including relatively minor amountsof dextrose, sodium chloride and potassium chloride. The buffer solutionshould be isotonic, with a pH of about 7. The BSA protein serves todecrease interactions which tend to interfere with the analysis. Thetarget substance may be added to the test medium before, after orsimultaneously with introduction of the magnetic particles. The methodof the invention takes advantage of the diffusion controlled solutionkinetics of the colloidal magnetic particles, which may be furtherenhanced by the addition of heat to the test medium. The test medium isusually incubated to promote binding between the receptor and any ligandof interest present therein. Incubation is typically conducted at roomtemperature or at a temperature slightly above the freezing point of thetest medium (i.e., 4° C. in an aqueous medium). The period of incubationis normally of short duration (i.e., about 15 minutes). The test mediummay be agitated or stirred during the incubation period to facilitatecontact between receptors and ligands.

Referring to FIG. 1, there is shown an exploded view of a magneticimmobilization apparatus 20a. A non-magnetic vessel, such as vessel 22ais provided with a central trough 21a and end pieces 23a and 23b forcontaining a fluid test medium (not shown). The vessel 22a is situatedbetween a north pole 24 and a south pole 25 of a device for generating amagnetic field transverse to the longitudinal axis of the vessel 22a.The devices 24 and 25, shown diagrammatically in FIG. 1, may be polepieces of an electromagnet, two confronting permanent magnets, or partsof a single channel, or a U-shaped, permanent magnet. A ferromagneticflux intensifier, such as collection wire 28a, spans between the endpieces 23a and 23b within the vessel 22a with an orientationsubstantially transverse to the lines of magnetic flux between the poles24 and 25 respectively. A ferromagnetic wire, when used as thecollection structure 28a, provides localized intensification of themagnetic flux density commensurate with the curvature of the wire. Thetwo ends of the wire 28a are attached to and are supported between theinterior surfaces of the end pieces 23a and 23b so that the wire spansthe interior volume of the vessel co-axially with the longitudinal axisof the wire. The two ends of the wire may be supported by, or embeddedwithin the end pieces such that the wire is substantiaiiy straight alongthe entire length thereof between the end pieces. The wire 28a may havea non-magnetic coating to reduce adhesion of magnetically-responsiveparticles in the absence of a magnetic field.

The immobilization apparatus 20a may be mounted on a stage (not shown)having micromanipulation devices and a suitable microscope to allow anoperator to manipulate and/or observe the immobilized target entities.The substantially linear form of the collection surfaces facilitatesmicroscopic observation of the immobilized entities along the length ofthe collection structure since such a linear array can easily bemaintained within the focal plane of a microscope. Direct observation ofthe immobilized entities can be made along an unobscured optical pathextending into the interior of the vessel and intersecting thecollection surfaces along either side of the collection structure. Thevessel 22a, as shown in FIG. 1, has an open top to facilitate access tothe interior by such devices as may be used in the practice of cellmicrosurgery or other biotechnical or chemical investigations.

Collection of target entities upon the surface of the collection wire28a is dominant along the wire surface regions most nearly orthogonal tothe lines of magnetic flux, i.e. along the opposite sides 39a and 39a'of the wire 28a which face the poles 24 and 25.

In applications wherein it is not desirable for physical contact tooccur between the target entities and a metallic surface, a non-metalliccoating may be applied to the surface of the ferromagnetic collectionstructure. When such a coating is present upon the ferromagneticcollection structure, the target entities are collected upon acollection surface that is upon the non-metallic coating and coextensivewith the ferromagnetic collection structure.

When the diameter of the collector wire 28a is chosen such that thecurvature of the wire 28a approximates the curvature of the targetentity, linear monolayers of the target entity, or one-dimensionalarrays, tend to form upon the collection wire 28a. As target entitiessuch as cells collect upon the wire 28a, a cell which collides withanother cell already attached to the wire 28a will tend to roll over theattached cell and then attach to an adjacent section of exposed wire.When a cell collides between two adjacent cells attached to the wire28a, the two attached cells tend to move apart to accommodate attachmentof the newly arrived cell to the wire. For example, it has been foundthat lymphocytes having a diameter on the order of 10 μm will tend tocollect in linear monolayer arrays upon either side of a 0.02 mm wirefrom solutions wherein the number of lymphocytes is limiting. Suchhighly ordered collection of target entities facilitates observation aswell as imaging by automatic processes.

The diameter of the collection wire may be selected in accordance withthe diameter of the target entities in order to produce a linearmonolayer of immobilized entities. In some instances, the diameter ofthe target entities may be such that a corresponding cylindricalcollection wire would not have enough bulk to exert a long-rangeinfluence upon the magnetic field within the vessel to attract thetarget entities toward the collection surface. In such instances, it maybe preferable to employ a collection structure having sufficient bulk toproduce a long-range magnetic gradient and also having a high curvaturesurface facing at least one of the magnetic pole faces. The highcurvature surface serves to produce a short-range gradient in thevicinity of the collection surface to arrange the entities that havebeen attracted by the long-range gradient into a linear monolayer uponthe collection surface. Such a collection structure may provide enhancedmechanical strength in addition to anisotropic intensification of themagnetic flux density. Such a structure is shown in FIG. 2A in the formof a wire 28b. The wire 28b has an elliptical, or ribbon-like, crosssection. When the wire 28b is positioned with the major elliptical axisaligned with the magnetic flux vector of an applied magnetic field, alarger degree of flux intensification is caused along the oppositesurfaces 39b and 39b' of the wire 28b having the highest curvature thanalong surfaces having lower curvature.

Another embodiment of a ferromagnetic collection structure for causinganisotropic flux intensification is shown in FIG. 2B. A composite wire,such as wire 28c, has a central strand 30 with two smaller strands 29parallel to the central strand 30 and positioned along opposite sides39c and 39c' of the central strand 30. A non-magnetic coating 31provides support for the strand assembly and allows for ease of removalof magnetically-responsive particles in the absence of a magnetic field.When the wire 28c is placed in a magnetic field such that the lines ofmagnetic flux are parallel to a line defined between the centers of thesmaller strands 29, maximum flux intensification is caused along thesurface regions 39c and 39c' of the wire 28c which are nearest to theexterior surfaces of the smaller strands 29. Other wire geometries orconfigurations may be employed within the scope of the present inventionin order to provide a high curvature surface while providing a greateramount of bulk material than a corresponding cylindrical wire. Suchcollection structures as are exemplified in FIGS. 2A-2B can be utilizedwithin any of the several immobilization vessels described and shownherein.

Alternative vessel structures may be used to provide enhanced spatialselectivity and mechanical support of target entity collection. In FIG.3A there is shown a central trough 21b for such a vessel. The centraltrough 21b includes a ferromagnetic collection structure in the form ofa wire 28d which is partially embedded in an interior lateral surface ofthe trough 21b. Collection of target entities in the central trough 21bwill predominate along the exposed portion 39d of the surface ofcollection wire 28d where such entities may easily be observed.Referring to FIG. 3B, there is shown a central trough 21c having acollection structure in the form of a wire 28e embedded entirely beneathan interior lateral surface 39e of the trough 21c. Collection of targetentities in a trough such as trough 21c will predominate along theinterior lateral surface 39e of the trough 21c in a line parallel to thewire 28e. Such an arrangement as trough 21c provides a substantiallyflat collection surface and minimal visual obscuration of the targetentities. Within the scope of the present invention, other geometricconfigurations of attached or embedded collection wires, such asrectilinear grids may be provided to produce a different pattern oftarget entity collection on the collection structure or on the interiorsurface of the trough.

Referring now to FIG. 3C, there is shown a central trough 21d having aflat, narrow collection structure, such as a razor blade 28f, embeddedwithin a side of the trough 21d with the edge 39f of the bladeprotruding into the interior of the trough and having a flat sidesubstantially parallel to the bottom of the trough 21d. The edge 39f ofcollection blade 28f may have a significantly sharper or greatercurvature than would be practical in embodiments employing wiressuspended within a trough. Such thin wires would be fragile and unlikelyto withstand magnetic field variations without substantial deformationThe angular edge 39f of collection element 28f has a high curvaturewhich provides a high magnetic field gradient in the vicinity of theprotruding edge thus promoting collection of target entities in a linearmonolayer.

A vessel such as is shown in FIG. 3D is preferable for such applicationsin which a linear monolayer of target entities is desired, but wherecontact with the edge of the collection element would damage the targetentities or be otherwise undesirable. The central trough 21e has a flat,narrow collection element 28g embedded within a side of the trough 2lein such a way that the edge of element 28g does not protrude into theinterior of the trough. In this case, the target entities are collectedon the interior lateral surface 39g along a line adjacent the edge ofthe element 28g.

Each of the trough structures described in connection with FIGS. 3A-3Dmay be provided with a transparent top member (not shown) or may remainopen when used in an immobilization apparatus in order to provide anunobstructed observation path intersecting the collection surface or toprovide direct physical access to the immobilized entities as may bedesired. Additionally, the central troughs 21b-21e may be partially orentirely transparent to further facilitate such observation.

It is often desired to expose target entities to various chemical andbiological environments other than the test medium in which such targetentities were originally suspended. It is also often desired to rinse orflush the test medium in which the target entities have been immobilizedin order to facilitate observation of the entities. Observation of theentities may be facilitated by flushing by allowing removal of an opaquemedium and by allowing control of separation time so that exposure tothe magnetic field may be limited to a duration sufficient for monolayerformation. A vessel which allows such environmental variation andflushing is shown in FIG. 1B. A flowthrough vessel for allowingalteration of the test environment, such as flowthrough vessel 22b, hasa central trough 21f which may be of such types as troughs 21a-ediscussed in connection with FIGS. 1, FIGS. 3A-3D, and FIG. 5B. Attachedto the ends of the central trough 21f are end pieces 23c, 23d having oneor more pairs of inlet and outlet ports such as ports 48a and 48b. Theend pieces 23c, 23d also provide support for a collection wire (notshown) in embodiments employing a wire spanning between the end pieces.The ports 48a and 48b are connected with hoses 46a, 46b which may befurther connected to sources of reagents, pumps, valves, and otherreagent flow control devices.

The flowthrough vessel 22b is provided with a top 40, which may betransparent to provide a clear optical path for microscopicobservations. For such observations wherein visible light or otherelectromagnetic radiation is observed after transmission through thesubject of interest, the bottom 41 of the central trough 21f may also betransparent to allow the passage of light through the vessel. Inalternative embodiments, the top 40 or any other parts of the vessel maybe selectively transparent to portions of the electromagnetic spectrumas may be desired in photobiological investigations. In still otherembodiments, the top 40 and the bottom 41 of the central trough 21f mayform a polarizer/analyzer pair for such applications as optical studiesof the mechanical properties of transparent membranes.

The flowthrough vessel 22b allows sequential exposure to reagents, suchas is required by indirect fluorescent MAb labelling, whilesignificantly reducing the loss of target entities as is usual intraditional methods which necessitate multiple, washings andcentrifugations. The flowthrough vessel 22b further allows for theconsumption of a reduced volume of reagents in such sequential processessince target entities are collected in a relatively high concentrationand with reduced spatial extent relative to known separation devices.The hoses 46a, 46b may be connected to a reagent recirculation systemfor further economy of reagent consumption.

A preferred manner of constructing an immobilization vessel isillustrated in FIG. 1C. The vessel 50 includes an upper assembly 52 anda lower assembly 54 which mate along surfaces 64 and 64' to form asubstantially rectangular hollow enclosure. The upper assembly 52 andthe lower assembly 54 are both transparent and have substantially flatexterior surfaces so that light may pass through the vessel 50 along asubstantially unobstructed and non-distorting optical path. Opposed ends53a and 53b of the lower assembly 54 have notches or grooves 56a and 56bformed along respective upper edges of the ends 53a and 53b. Before theupper assembly is secured to the lower assembly, a ferromagneticcollection structure 58 may be aligned within grooves 56a and 56b andsecured therein by an adhesive (not shown). Additional adhesive isapplied to the mating surfaces 64 and 64' of the upper and lowerassemblies and the mating surfaces are brought into contact with eachother. After the adhesive has set, the protruding portions of thecollection structure 58 may be trimmed flush with the exterior of thevessel.

In order to provide a flowthrough vessel, semicircular notches 60a,60a', and 60b may be formed the opposed ends of the upper and lowerassemblies along the mating surfaces 64, 64'. When the upper and lowerassemblies are secured together, inlet port nipple 62a and outlet portnipple 62b may be positioned so that the nipples 62a and 62b are securedwithin the mated semicircular notches. The flow path is through thehollow inlet nipple 62a, the hollow interior of the enclosure formed bythe assemblies 52 and 54, and the hollow outlet nipple 62b. Othermethods of assembling an immobilization apparatus, such as injectionmolding, are contemplated within the scope of the invention.

Turning now to FIG. 5, there is shown an exemplary arrangement in whichobservations of immobilized target entities can be made. A hollow vessel70, which may be of any type discussed herein, is supported upon amicroscope stage 72. The microscope stage 72 is equipped with awell-known mechanism for translating the vessel 70 in either directionalong the axis 73. Magnets 74 and 76 are positioned on either side ofthe microscope stage to establish an applied magnetic field transverseto the longitudinal axis of the vessel 70. A source of light 78 isprovided for projecting a collimated beam 80 of light toward the vessel70 along an axis that is perpendicular to the longitudinal axis of thevessel and to the applied magnetic field. The vessel 70 has a top and abottom that are transparent to the beam 80 of light. The top and bottomsurfaces of the vessel 70 are substantially flat so that the beam 80 maypass through the top and bottom surfaces substantially undistorted. Aferromagnetic collection structure 82 is positioned within the vessel 70and in the present instance is supported co-axially of the longitudinalaxis of the vessel 70. The ferromagnetic collection structure 82 has acoextensive collection surface upon which magnetically-labeled entitiesare adhered. The collection surface of the collection structure 82 isoriented relative to the path of beam 80 such that the optical path ofthe beam 80 intersects the collection surface so that the immobilizedentities may be visually observed. Such visual observation may be made,for example, by collecting the light transmitted through the vessel 70with an objective lens 84 and reflecting the collected light with amirror 86 toward an eyepiece 88.

Lateral translation of target entities along the collection surface maybe accomplished within an immobilization apparatus without necessitatingphysical contact between the target entities and a device such as amicromanipulator. A magnetic field generated between two confrontingpoles of finite lateral extent possesses a positive lateral gradienttoward the region between the poles. Hence, magnetically-labeled targetentities which have been collected on a ferromagnetic collectingstructure such as the wire 28a shown in FIG. 1A will tend to remainbetween the poles 24 and 25 as the vessel 22a is translated in adirection parallel to the longitudinal axis of the wire 28a. Therelative motion between a ferromagnetic collecting element and a targetentity generated in such a fashion may be used to position a targetentity at a selected location upon the surface of the ferromagneticcollecting element.

Lateral translation of target entities along the collection surface mayalso be employed to increase the local concentration of target entitieswhich have been collected. Referring to FIG. 4A, an alternativecollection structure is shown as a collection wire 28h having a reduceddiameter at 34 and an enlarged diameter at 35 interconnected by atransverse wall 36 to produce a shoulder therebetween. The collectionwire 28h is positioned between confronting opposite magnetic poles 24and 25 within a vessel (not shown) containing a fluid medium in whichare suspended magnetically-labeled target entities. The confinement ofmagnetic flux lines between poles 24 and 25 will cause collection oftarget entities along the portion of the outer sides of the wire 28e inthe reduced diameter portion 34 which is disposed directly between thepoles 24 and 25. The vessel supporting the wire 28h is then laterallytranslated, such as by translation of a supporting microscope stage (notshown), in the direction indicated by the arrow 38h so as to move theenlarged portion 35 of the wire 28h into the region between the poles 24and 25. The tendency of magnetically-responsive target entities toremain in the region of greatest magnetic flux density will cause thetarget entities to congregate on the portions of the collection surfaces39h and 39h' adjacent the opposite shoulders 37 and 37' between theanterior wall surface 36 of the enlarged portion 35 and the oppositesides 39h, 39h' of the reduced-diameter wire portion 34 facing the poles24 and 25.

Lateral translation and congregation of the target entities may also beaccomplished using vessels having embedded ferromagnetic collectionstructures similar to those shown in central troughs 21b-21e shown inFIGS. 3A-3D. Referring to FIG. 4B, a central trough 21i having anembedded ferromagnetic collection element 28i is shown. The centraltrough 21i further includes a surface irregularity, such as a transverseobstacle or weir 48, forming a shoulder on the interior surface 39ilocated along the line of collection. As the central trough 21i islaterally translated in the direction indicated by the arrow 38i, targetentities collected upon the interior surface 39i of the central troughwill tend to congregate at the angular junction between the line ofcollection and the anterior surface 49 of the shoulder 48. The abilityto create dense congregations of target entities without directmechanical interventions provides unique advantages for suchapplications as in vitro fertilization or other techniques in which itis desired to propagate a culture of fragile target entities whichreproduce at a rate commensurate with their concentration.

The following examples further describe in some detail manners of usingaspects of the present invention and set forth the best modecontemplated by the inventors for carrying out the invention, but arenot to be construed as limiting the invention.

EXAMPLE 1

To demonstrate the feasibility of one-dimensional immobilization ofcells within a test medium, an immobilization apparatus using a centraltrough of the type shown in FIG. 3C was constructed and tested.Permanent rare earth alloy magnets made of CRUMAX 355 (a trademark ofCrucible Magnetics of Elizabethtown, Ky.) were used to generate themagnetic field. The magnets were in the form of bar magnets havingdimensions of 1.75 inches×0.375 inches×0.375 inches with a flux vectorparallel to one of the 0.375 dimensions. The magnets were mounted in ayoke made of cold rolled steel. The opposing faces of the magnets were0.3125 inches apart. The magnetic field strength at each bar magnet facewas measured to be 5.8 kGauss. The vessel had outer dimensions of 0.1875inches×0.3125 inches×0.5 inches with the 0.3125 inch dimension fittedbetween the magnet faces. The wall thickness of the vessel was 0.0625in. The ferromagnetic collection element was a portion of a razor blade.

A dextran-coated magnetic colloid was prepared having a concentration of0.0212 μg/ml of Fe. Five ml of the magnetic colloid was incubated withcells of the T-Cell CEM line. The colloid coated the cells vianon-specific binding. The final concentration of T-Cells in the mixturewas 2,000 cells/ml. Next, 50 μl aliquots of the cell mixture wereinserted into the immobilization vessel and allowed to separate for 5minutes. Excess cells were flushed away with PBS buffer. A single lineof cells collected along the edge of the razor blade were observed withan optical microscope at 30× magnification.

EXAMPLE 2

To demonstrate chemical manipulation of cells subsequent toimmobilization, T-Cells were first immobilized as in Example 1 above.After collection, the cells were washed three times in the usual mannerwith PBS buffer to remove excess magnetic particles from the mixture.Then, 12 μl of Fitc CD45 T-Cell marker having a concentration of 10μg/ml was added to the cell mixture and incubated for five minutes atroom temperature. After the labelling step, the cells were washed threetimes again in PBS buffer. Fluorescently-labelled cells were observed tobe aligned with the edge of the razor blade via 60× magnificationfluorescent microscopy.

EXAMPLE 3

To demonstrate micromanipulation of cells subsequent to immobilization,2,000 magnetically labeled T-Cells in 20 μl of 10 mM PBS buffer wereinjected into the immobilization vessel and separated as in Example 1.The cells were subsequently washed with PBS buffer to remove any unboundcells or cellular debris. Acridine Orange was allowed to flow past theimmobilized cells. Using an optical microscope at 600× magnification,the cell nuclei appeared to glow a bright orange. 1591 immobilized cellswere counted optically. The cells were then allowed to incubate for aperiod of 5 hours. A secondary stain, DIL-282, available from MolecularProbes of Eugene, Oreg., was injected in to the chamber to selectivelystain the portions of the cell surfaces not in direct contact with therazor edge or neighboring cells. The cells were then washed twice in PBSbuffer. The immobilization vessel was taken out of the magnetic field toeffect re-orientation of the cells and agitated for one minute. Thevessel was then placed back into the magnetic field. Surface portions ofcells which had previously been in direct contact with neighboring cellssurfaces were visible and unstained. Sections of such unstained cellmembranes were surgically removed for a comparison of the chemicalcomposition of such sections with comparable sections of stained cellmembrane in order to identify chemical cellular communication agents.

From the foregoing disclosure and the accompanying drawings, it can beseen that the present invention provides certain novel and usefulfeatures that will be apparent to those skilled in the pertinent art. Inparticular there has been described an apparatus in which biologicalentities are biospecifically magnetically labeled and immobilized. Suchan apparatus provides rapid immobilization of target entities while theymay remain within a fluid medium thus retaining viability and avoidingpotentially damaging physical contact as occurs in techniques availablehitherto. The apparatus disclosed herein further facilitates observationand manipulation of the immobilized entities by manual and automatedtechniques. Various embodiments of the present invention allow arbitraryconfigurational capture, spatial translation, and sequential reagentexposure of the target entities.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described, or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed.

That which is claimed is:
 1. A method for observing microbiological entities, selected from a group consisting of cells or microbes, suspended in a fluid medium, the method comprising the steps of:rendering the entities magnetically responsive by contacting the entities with a plurality of magnetic particles bearing an attachment agent having a binding affinity for the entities; providing a vessel with a transparent wall and a ferromagnetic collection structure having a collection surface with a diameter approximately equal to or less than the diameter of the entities; introducing the fluid medium into the vessel; placing the vessel into a magnetic field for inducing a magnetic gradient about the collection structure of sufficient strength to immobilize the entities; attracting the entities toward the collection structure; collecting a substantially linear monolayer of the biological substance along the collection structure; and optically observing the linear monolayer through the transparent wall.
 2. The method of claim 1 wherein the collection structure is supported by and along an interior surface of the vessel, and said attracting step comprises the step of attracting the biological substance toward said interior surface along which the collection structure is supported.
 3. The method of claim 2, wherein the collection surface is coextensive with the interior surface.
 4. The method of claim 3 wherein the collection structure is at least partially embedded in the interior surface of the vessel supporting the collection structure.
 5. The method of claim 1 wherein the entities comprise leukocytes.
 6. The method of claim 5 wherein the rendering step comprises the step of contacting the leukocytes with a plurality of dextran-bearing magnetic particles.
 7. The method of claim 1 wherein said observing step comprises observing a fluorescence characteristic of the entities collected in a linear monolayer.
 8. The method of claim 7 wherein the entities comprise lymphocytes having a diameter on the order of 10 μm.
 9. The method of claim 1 wherein the entities comprise lymphocytes having a diameter on the order of 10 μm.
 10. The method of claim 1 wherein the collection structure is supported by and along an interior surface of the vessel, the interior surface is formed to have a shoulder, and said attracting step comprises the step of attracting the biological substance toward said interior surface, the method further comprising the step of mechanically manipulating the collected biological substance by translating the shoulder relative to the magnetic field.
 11. A method for observing a microscopic biological target comprising a cell membrane having a binding receptor and suspended in a fluid medium, comprising the steps of:rendering the substance magnetically responsive by contacting the target with a plurality of magnetically responsive particles having a binding ligand with an immunospecific binding affinity for the receptor; introducing the fluid medium into a vessel having a ferromagnetic collection structure and a transparent wall; placing the vessel into a magnetic field for inducing a magnetic gradient about the collection structure of sufficient strength to immobilize the biological substance; attracting the microscopic biological target toward the collection structure; collecting a substantially linear monolayer of the biological substance along the collection structure; and optically observing the linear monolayer through the transparent wall.
 12. The method of claim 11 wherein the collection structure is supported by and along an interior surface of the vessel, and said attracting step comprises the step of attracting the target toward said interior surface along which the collection structure is supported.
 13. The method of claim 12 wherein the collection surface of the collection structure is coextensive with the interior surface.
 14. The method of claim 13 wherein the collection structure is at least partially embedded in the interior surface of the vessel supporting the collection structure.
 15. The method of claim 11 wherein said observing step comprises observing a fluorescence characteristic of the target collected in a linear monolayer.
 16. The method of claim 11 wherein said collection stricture has a collection surface having a diameter approximately equal to a diameter of the biological substance.
 17. The method of claim 16 wherein the biological substance comprises lymphocytes having a diameter on the order of 10 μm.
 18. The method of claim 11 wherein the collection is supported structure along an interior surface of the vessel, the interior surface has a shoulder, and said attracting step comprises the step of attracting the biological substance toward said interior surface, the method further comprising the step of mechanically manipulating the collected biological substance by translating the shoulder relative to the magnetic field. 